We report the first small-molecule protease-activated receptor (PAR) 2 agonists, AC-55541 N-1-(3-bromo-phenyl)-eth-(E)-ylidene-hydrazinocarbonyl-(4-oxo-3,4-dihydro-phthalazin-1-yl)-methyl-benzamide ...and AC-264613 2-oxo-4-phenylpyrrolidine-3-carboxylic acid 1-(3-bromo-phenyl)-(E/Z)-ethylidene-hydrazide, each representing a distinct chemical series. AC-55541 and AC-264613 each activated PAR2 signaling in cellular proliferation assays, phosphatidylinositol hydrolysis assays, and Ca(2+) mobilization assays, with potencies ranging from 200 to 1000 nM for AC-55541 and 30 to 100 nM for AC-264613. In comparison, the PAR2-activating peptide 2-furoyl-LIGRLO-NH(2) had similar potency, whereas SLIGRL-NH(2) was 30 to 300 times less potent. Neither AC-55541 nor AC-264613 had activity at any of the other PAR receptor subtypes, nor did they have any significant affinity for over 30 other molecular targets involved in nociception. Visualization of EYFP-tagged PAR2 receptors showed that each compound stimulated internalization of PAR2 receptors. AC-55541 and AC-264613 were well absorbed when administered intraperitoneally to rats, each reaching micromolar peak plasma concentrations. AC-55541 and AC-264613 were each stable to metabolism by liver microsomes and maintained sustained exposure in rats, with elimination half-lives of 6.1 and 2.5 h, respectively. Intrapaw administration of AC-55541 or AC-264613 elicited robust and persistent thermal hyperalgesia and edema. Coadministration of either a tachykinin 1 (neurokinin 1) receptor antagonist or a transient receptor potential vanilloid (TRPV) 1 antagonist completely blocked these effects. Systemic administration of either AC-55541 or AC-264613 produced a similar degree of hyperalgesia as was observed when the compounds were administered locally. These compounds represent novel small-molecule PAR2 agonists that will be useful in probing the physiological functions of PAR2 receptors.
We expressed the cloned mu-opioid receptor (muR) in high abundance (5.5 x 10(6) sites/cell) with an amino-terminal epitope tag (EYMPME) in human embryonic kidney 293 cells. The epitope-tagged ...receptor (EE-muR) was similar to the untagged mu R ligand binding and agonist-dependent inhibition of cyclic AMP accumulation. By confocal microscopy, the labeled receptor was shown to be largely confined to the plasma membrane. Pretreatment with morphine failed to affect the cellular distribution of the receptor as judged by immunofluorescence and tracer binding studies. In contrast, exposure to the mu-specific peptide agonist D-Ala2, MePhe4, Gly-ol5enkephalin (DAMGO) caused strong labeling of endocytic vesicles, indicating extensive agonist-induced cellular redistribution of EE-muR. Tracer binding studies suggested partial net internalization and a small degree of down-regulation caused by DAMGO. EE-muR-containing membranes were solubilized in detergent 3-(3-cholamidopropyl) dimethylammonio-1-propanesulfonate and immunoprecipitated by an anti-epitope monoclonal antibody. Immunoblotting revealed a prominent band at approximately 70 kDa with weaker bands at approximately 65 kDa. EE-muR was labeled with gamma-32PATP in permeabilized cells, immunoprecipitated, and analyzed by polyacrylamide gel electrophoresis autoradiography. A prominent band at 65-70 kDa indicated the presence of basal receptor phosphorylation occurring in the absence of agonist, which was enhanced approximately 1.8-fold with the addition of morphine. In conclusion, intracellular trafficking of the muR appears to depend on the agonist, with morphine and DAMGO having markedly different effects. Unlike other G protein-coupled receptors, basal phosphorylation is substantial, even in the absence of agonist.
PD-1/L1 axis-directed therapies produce clinical responses in a subset of patients; therefore, biomarkers of response are needed. We hypothesized that quantifying key immunosuppression mechanisms ...within the tumor microenvironment by multiparameter algorithms would identify strong predictors of anti-PD-1 response.
Pretreatment tumor biopsies from 166 patients treated with anti-PD-1 across 10 academic cancer centers were fluorescently stained with multiple markers in discovery (
= 24) and validation (
= 142) cohorts. Biomarker-positive cells and their colocalization were spatially profiled in pathologist-selected tumor regions using novel Automated Quantitative Analysis algorithms. Selected biomarker signatures, PD-1/PD-L1 interaction score, and IDO-1/HLA-DR coexpression were evaluated for anti-PD-1 treatment outcomes.
In the discovery cohort, PD-1/PD-L1 interaction score and/or IDO-1/HLA-DR coexpression was strongly associated with anti-PD-1 response (
= 0.0005). In contrast, individual biomarkers (PD-1, PD-L1, IDO-1, HLA-DR) were not associated with response or survival. This finding was replicated in an independent validation cohort: patients with high PD-1/PD-L1 and/or IDO-1/HLA-DR were more likely to respond (
= 0.0096). These patients also experienced significantly improved progression-free survival (HR = 0.36;
= 0.0004) and overall survival (HR = 0.39;
= 0.0011). In the combined cohort, 80% of patients exhibiting higher levels of PD-1/PD-L1 interaction scores and IDO-1/HLA-DR responded to PD-1 blockers (
= 0.000004). In contrast, PD-L1 expression was not predictive of survival.
Quantitative spatial profiling of key tumor-immune suppression pathways by novel digital pathology algorithms could help more reliably select melanoma patients for PD-1 monotherapy.
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We assessed a simple, noninvasive method of monitoring transcutaneous partial pressure of CO2 (Ptcco2) in mice to determine whether it would provide an accurate and reproducible method to assess ...ventilatory depression in mice. To this end, Ptcco2 and Paco2 (partial pressure of arterial CO2) measurements were performed on isoflurane-anesthetized male C57Bl/6 mice breathing differing percentages of CO2 or fentanyl, a known ventilatory depressive drug. All doses of fentanyl produced a sharp increase in Ptcco2 values within 20 min with difference in Ptcco2 values between saline and all fentanyl groups being statistically significant (P < 0.0001). A good correlation between Paco2 and Ptcco2 values was established (r2 = 0.91). A Bland-Altman analysis likewise found that Ptcco2 measurements in the mice reliably and accurately reflected their Paco2 values. Therefore, under controlled conditions, Ptcco2 measurements were found to reliably reflect Paco2 values in mice. Consequently, the Ptcco2 method can be used as a means to rapidly and quantitatively assess the ventilatory depressive properties of a wide spectrum of drugs, under varying conditions in numerous mouse models.
The mechanism by which muscarinic receptors internalize upon agonist exposure is poorly understood. To determine the endocytic pathways responsible for muscarinic receptor internalization, we have ...stably transfected human embryonic kidney (HEK 293) cells with the Hm1 (human muscarinic subtype 1) receptor tagged at the amino terminus with the epitope EYMPME. The subcellular location of the receptor was visualized by immunofluorescence confocal microscopy and quantified with the use of binding studies. The receptor redistributed into intracellular compartments following agonist treatment. This process was reversible upon removal of agonist and inhibited by antagonist. Acid treatment of the cells, which disrupts internalization via clathrin-coated vesicles, inhibited carbachol-stimulated internalization. Phorbol 12-myristate 13-acetate, on the other hand, which inhibits caveolae-mediated endocytosis, had no effect on carbachol-induced endocytosis. Double-labeling confocal microscopy was used to characterize the intracellular vesicles containing Hm1 receptor following agonist treatment. The Hm1 receptor was shown to be colocalized with clathrin and α-adaptin, a subunit of the AP2 adaptor protein which links endocytosed proteins with clathrin in the intracellular vesicles. In addition, endosomes containing Hm1 also contained the transferrin receptor, which internalizes via clathrin-coated vesicles. In contrast, caveolin, the protein that comprises caveolae, did not colocalize with Hm1 in intracellular vesicles following agonist treatment, indicating that caveolae are not involved in the agonist-induced internalization of Hm1. These results indicate that agonist-induced internalization of the Hm1 receptor occurs via clathrin-coated vesicles in HEK cells.
The aim of the present study was to describe the activity of a set of opioid drugs, including partial agonists, in a human embryonic kidney cell system stably expressing only the mouse kappa-opioid ...receptors. Receptor activation was assessed by measuring the inhibition of cyclic adenosine mono phosphate (cAMP) production stimulated by 5 microM forskolin. Intrinsic activities and potencies of these ligands were determined relative to the endogenous ligand dynorphin and the kappa agonist with the highest intrinsic activity that was identified in this study, fentanyl.
Among the ligands studied naltrexone, WIN 44,441 and dezocine, were classified as antagonists, while the remaining ligands were agonists. Intrinsic activity of agonists was assessed by determining the extent of inhibition of forskolin-stimulated cAMP production. The absolute levels of inhibition of cAMP production by each ligand was used to describe the rank order of intrinsic activity of the agonists; fentanyl = lofentanil > or = hydromorphone = morphine = nalorphine > or = etorphine > or = xorphanol > or = metazocine > or = SKF 10047 = cyclazocine > or = butorphanol > nalbuphine. The rank order of affinity of these ligands was; cyclazocine > naltrexone > or = SKF 10047 > or = xorphanol > or = WIN 44,441 > nalorphine > butorphanol > nalbuphine > or = lofentanil > dezocine > or = metazocine > or = morphine > hydromorphone > fentanyl.
These results elucidate the relative activities of a set of opioid ligands at kappa-opioid receptor and can serve as the initial step in a systematic study leading to understanding of the mode of action of these opioid ligands at this receptor.
The aim of the present study was to characterize the activation profiles of 15 opioid ligands in transfected human embryonic kidney cells expressing only delta opioid receptors. Activation profiles ...of most of these ligands at delta opioid receptors had not been previously characterized in vitro. Receptor activation was assessed by measuring the inhibition of forskolin-stimulated cAMP production.
Naltrexone and nalorphine were classified as antagonists at delta opioid receptor. The other ligands studied were agonists at delta opioid receptors and demonstrated IC50 values of 0.1 nM to 2 microM, maximal inhibition of 39-77% and receptor binding affinities of 0.5 to 243 nM. The rank order of efficacy of the ligands tested was metazocine = xorphanol > or = fentanyl = SKF 10047 = etorphine = hydromorphone = butorphanol = lofentanil > WIN 44,441 = Nalbuphine = cyclazocine > or = met-enkephalin >> morphine = dezocine. For the first time these data describe and compare the function and relative efficacy of several ligands at delta opioid receptors.
The data produced from this study can lead to elucidation of the complete activation profiles of several opioid ligands, leading to clarification of the mechanisms involved in physiological effects of these ligands at delta opioid receptors. Furthermore, these data can be used as a basis for novel use of existing opioid ligands based on their pharmacology at delta opioid receptors.
Introduction
Acute myeloid leukemia (AML) is a heterogeneous disease with multiple factors influencing long-term outcome. FLT3 (FMS-like tyrosine kinase 3) mutations (predominantly internal tandem ...duplication ITD) are reported in ~25% of patients with AML and have been associated with poorer outcomes compared with FLT3 non-mutated AML (Kottaridis et al. Blood. 2001). Targeting these mutations with tyrosine kinase inhibitors has become an active area of research. However, patients must undergo molecular testing to identify the presence of this mutation. The specimen of choice for such testing is bone marrow (BM; aspirate or biopsy), as the site of origin of the disease. However, BM testing is invasive and sometimes cannot be performed repeatedly in a timely manner. A simple technique using easily obtainable biological specimens from a source such as peripheral blood (PB) may be desirable if the results accurately reflect status in the active disease site (i.e., BM). We investigated whether a PB specimen could be utilized for FLT3 -ITD determination and if the characteristics of the ITD mutations identified in PB were consistent with those in BM samples from the same patient.
Methods
QuANTUM-R is a global, randomized, phase 3 study to assess the efficacy of quizartinib, a selective FLT3 inhibitor, in patients with FLT3 -ITD-mutated relapsed or refractory (R/R) AML. Paired samples of BM and PB were collected from individual patients as part of the screening process for QuANTUM-R. One to 3 mL each of BM and PB were collected on the same day (window Day -14 to Day 0) from each patient prior to receipt of any study treatment. A statistically powered number of patients (minimum 95) with paired BM and PB samples were selected during a defined period of screening in the phase 3 study. Samples were subjected to DNA isolation followed by PCR and fragment size analysis. Each sample was assessed for the presence of ITD mutation; moreover, the length and allelic frequency of the ITD mutation were measured for each sample. The assay was validated down to a limit of detection (LoD) of 1%. Correlation between the data obtained from BM and PB was assessed using Deming regression analysis and Bland-Altman plot constructed to assess concordance between the 2 sample types.
Results
Paired samples from 107 patients (median age 58y; 32% were ≥65y; 57% were female) were included. Sixteen samples had no ITD mutations in either BM or PB; 3 had ITD only in the BM sample with no corresponding ITD in the paired PB sample (despite having circulating blasts), and 88 had ITD in both BM and PB. In all 88 cases, the length of the ITD was identical in both specimen types. The sizes of ITD observed in this patient population were between 9 and 198 bases; 21/88 samples with ITD demonstrated the presence of multiple ITDs, with samples from 2 patients exhibiting 3 distinct ITDs, each of whose sizes were the same in BM and PB. Across all samples with ITD, the measured ITD allelic ratio ranged from 1% to 96%, with a similar or slightly lower ratio measured in the PB compared to the BM. In 23 (26%) cases, the allelic ratio measured in the PB was lower than that of the BM by ≥10%. Blast counts for these samples were also measured and there was no correlation between blast count and allelic ratio in either specimen type. High correlation between PB and BM samples for the presence of FLT3 -ITD was observed when regressing PB Ratio on BM Ratio via Deming regression analysis, and the following values were obtained: R2 = 0.866; 95% CI for the slope, 0.98 to 1.12; and Bland-Altman difference plot for allelic ratio against difference between PB and BM showed good random scatter around average bias of -5.2%, with 95% CI including 0.0% (95% CI, -27.6 to 17.2).
Conclusions
FLT3 -ITD mutation testing is performed in all AML patients at diagnosis for prognostic purposes and to guide therapeutic decisions. As FLT3 inhibitors are developed for clinical use, regular monitoring of patients' residual disease burden/response to therapy through assessment of FLT3 status may become an important element of monitoring benefit from effective therapy. Mutation assessment is currently performed on BM aspirate or biopsy based on the common belief that PB is not adequate for this assessment. Our results of FLT3 -ITD mutation analysis in paired BM and PB samples collected from >100 patients with R/R AML show that PB specimens have a high degree of concordance with BM specimens for assessment of FLT3 mutation while being less invasive.
Khaled:Daiichi Sankyo, Inc: Other: Travel Support; City of Hope: Research Funding. Ganguly:Amgen: Other: Advisory Board; Seattle Genetics: Speakers Bureau. Perl:Seattle Genetics: Other: Advisory board; Daiichi Sankyo: Consultancy; Astellas: Consultancy; Novartis: Other: Advisory Board; Pfizer: Other: Advisory Board; Actinium Pharmaceuticals: Other: Scientific Advisory Board; Arog Pharmaceuticals: Consultancy; Asana Biosciences: Other: Scientific advisory board. Kobayashi:Daiichi Sankyo, Inc.: Employment. Berisha:Daiichi Sankyo, Inc.: Employment. Lameh:Daiichi Sankyo, Inc.: Research Funding; Navigate BioPharma Service Inc.,: Employment. Martinelli:Celgene: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Ariad: Consultancy; Incyte-Teva: Speakers Bureau.
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